Sonkar Amit, Shukla Harish, Shukla Rohit, Kalita Jupitara, Pandey Tripti, Tripathi Timir
Molecular and Structural Biophysics Laboratory, Department of Biochemistry, North-Eastern Hill University, Shillong 793022, India.
Molecular and Structural Biophysics Laboratory, Department of Biochemistry, North-Eastern Hill University, Shillong 793022, India.
Int J Biol Macromol. 2017 Apr;97:106-114. doi: 10.1016/j.ijbiomac.2016.12.082. Epub 2017 Jan 4.
Peptidoglycan (PG) is the key component of the bacterial cell wall. The enzyme UDP-N-Acetylglucosamine Enolpyruvyl Transferase (MurA) catalyzes the transfer of enolpyruvate from phosphoenolpyruvate (PEP) to uridinediphospho-N-acetylglucosamine (UNAG), which is the first committed step of PG biosynthesis. Here, we present the biochemical and structural features of the MurA enzyme of the opportunistic pathogen Acinetobacter baumannii (AbMurA). The recombinant AbMurA exists as a monomer in solution and shows optimal activity at pH 7.5 and 37°C. The Km for UDP-N-acetylglucosamine was 1.062±0.09mM and for PEP was 1.806±0.23mM. The relative enzymatic activity was inhibited ∼3 fold in the presence of 50mM fosfomycin (FFQ). Superimposition of the AbMurA model with E. coli demonstrated key structural similarity in the FFQ-binding site. AbMurA also has a surface loop that contains the active site Cys116 that interact with FFQ. Sequence analysis indicates the presence of the five conserved amino acids, i.e., K22, C116, D306, D370 and L371, required for the functional activity like other MurA enzymes from different bacteria. MurA enzymes are indispensable for cell integrity and their lack of counterparts in eukaryotes suggests them to be a promising drug target.
肽聚糖（PG）是细菌细胞壁的关键成分。UDP-N-乙酰葡糖胺烯醇丙酮酸转移酶（MurA）催化烯醇丙酮酸从磷酸烯醇丙酮酸（PEP）转移至尿苷二磷酸-N-乙酰葡糖胺（UNAG），这是PG生物合成的首个关键步骤。在此，我们展示了机会致病菌鲍曼不动杆菌（AbMurA）的MurA酶的生化及结构特征。重组AbMurA在溶液中以单体形式存在，在pH 7.5和37°C时表现出最佳活性。UDP-N-乙酰葡糖胺的Km为1.062±0.09mM，PEP的Km为1.806±0.23mM。在50mM磷霉素（FFQ）存在的情况下，相对酶活性受到约3倍的抑制。将AbMurA模型与大肠杆菌进行叠加显示，在FFQ结合位点存在关键的结构相似性。AbMurA还有一个表面环，其中包含与FFQ相互作用的活性位点Cys116。序列分析表明，存在五个保守氨基酸，即K22、C116、D306、D370和L371，它们是与来自不同细菌的其他MurA酶一样具有功能活性所必需的。MurA酶对于细胞完整性不可或缺，且在真核生物中不存在对应物，这表明它们是有前景的药物靶点。
